CRISPR Journal最新文献

{"title":"CRISPR-Cas9 Long-Read Sequencing for Mapping Transgenes in the Mouse Genome.","authors":"W Bart Bryant, Allison Yang, Susan H Griffin, Wei Zhang, Ashiq M Rafiq, Weiping Han, Ferenc Deak, Mary Katherine Mills, Xiaochun Long, Joseph M Miano","doi":"10.1089/crispr.2022.0099","DOIUrl":"10.1089/crispr.2022.0099","url":null,"abstract":"<p><p>Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci. This novel approach mapped a wide size range of transgenes and uncovered more complex transgene-induced host genome re-arrangements than previously appreciated. CRISPR-LRS offers a facile, informative approach to establish robust breeding practices and will enable researchers to study a gene without confounding genetic issues. Finally, CRISPR-LRS will find utility in rapidly and accurately interrogating gene/genome editing fidelity in experimental and clinical settings.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123806/pdf/crispr.2022.0099.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10187974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Amplifying CRISPR: Next-Generation Diagnostics.","authors":"Rodolphe Barrangou","doi":"10.1089/crispr.2023.0004.editorial","DOIUrl":"https://doi.org/10.1089/crispr.2023.0004.editorial","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9686846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Periodontal Disease Pathogens, Pathogenesis, and Therapeutics: The CRISPR-Cas Effect.","authors":"Madhurya N Kedlaya,&nbsp;Lakshmi Puzhankara,&nbsp;Rohit Prasad,&nbsp;Akshatha Raj","doi":"10.1089/crispr.2022.0094","DOIUrl":"https://doi.org/10.1089/crispr.2022.0094","url":null,"abstract":"<p><p>Periodontal disease (PD) is an immune-inflammatory disease affecting the supporting structures of the teeth, which results in progressive destruction of the hard and soft tissues surrounding teeth, ultimately resulting in tooth loss. The primary etiological factor for this disease is the presence of pathogenic microorganisms. Pathogenic bacteria face antagonistic conditions and foreign DNA components during the infection stage and depend on defense mechanisms such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas to counter them. Virulence genes regulated by the CRISPR-Cas system are often expressed by bacteria as part of the stress response to the presence of stress conditions and foreign elements. There is ever-growing evidence regarding the role of CRISPR-Cas in virulence of periodontal pathogens. The same CRISPR-Cas system may also be targeted to reduce bacterial virulence and it may also be utilized to develop diagnostic and therapeutic strategies for prevention and control of PD progression. This review article describes the CRISPR-Cas systems in the periodontal dysbiotic microbial communities, their role in the virulence of periodontal pathogens, and their potential role in understanding the pathogenesis of periodontitis and treatment of PD.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9685226","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Functional and Phylogenetic Diversity of Cas10 Proteins.","authors":"Tanner Wiegand, Royce Wilkinson, Andrew Santiago-Frangos, Mackenzie Lynes, Roland Hatzenpichler, Blake Wiedenheft","doi":"10.1089/crispr.2022.0085","DOIUrl":"10.1089/crispr.2022.0085","url":null,"abstract":"<p><p>Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10123807/pdf/crispr.2022.0085.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9472342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.","authors":"Maturada Patchsung,&nbsp;Aimorn Homchan,&nbsp;Kanokpol Aphicho,&nbsp;Surased Suraritdechachai,&nbsp;Thanyapat Wanitchanon,&nbsp;Archiraya Pattama,&nbsp;Khomkrit Sappakhaw,&nbsp;Piyachat Meesawat,&nbsp;Thanakrit Wongsatit,&nbsp;Artittaya Athipanyasilp,&nbsp;Krittapas Jantarug,&nbsp;Niracha Athipanyasilp,&nbsp;Juthamas Buahom,&nbsp;Supapat Visanpattanasin,&nbsp;Nootaree Niljianskul,&nbsp;Pimchai Chaiyen,&nbsp;Ruchanok Tinikul,&nbsp;Nuanjun Wichukchinda,&nbsp;Surakameth Mahasirimongkol,&nbsp;Rujipas Sirijatuphat,&nbsp;Nasikarn Angkasekwinai,&nbsp;Michael A Crone,&nbsp;Paul S Freemont,&nbsp;Julia Joung,&nbsp;Alim Ladha,&nbsp;Omar Abudayyeh,&nbsp;Jonathan Gootenberg,&nbsp;Feng Zhang,&nbsp;Claire Chewapreecha,&nbsp;Sittinan Chanarat,&nbsp;Navin Horthongkham,&nbsp;Danaya Pakotiprapha,&nbsp;Chayasith Uttamapinant","doi":"10.1089/crispr.2022.0048","DOIUrl":"https://doi.org/10.1089/crispr.2022.0048","url":null,"abstract":"<p><p>Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7614457/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9384149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiplex gRNAs Synergically Enhance Detection of SARS-CoV-2 by CRISPR-Cas12a.","authors":"Melissa D Morales-Moreno, Erick G Valdés-Galindo, Mariana M Reza, Tatiana Fiordelisio, Jorge Peon, Armando Hernandez-Garcia","doi":"10.1089/crispr.2022.0074","DOIUrl":"10.1089/crispr.2022.0074","url":null,"abstract":"<p><p>Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in ∼25 min using a fluorescence plate reader and ∼45 min with lateral flow strips.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9330781","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Special Issue</i>: Manipulating the Microbiome with CRISPR.","authors":"Brady Cress,&nbsp;Rodolphe Barrangou","doi":"10.1089/crispr.2023.0005.cfp","DOIUrl":"https://doi.org/10.1089/crispr.2023.0005.cfp","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9686845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic <i>BCR::ABL1</i> and <i>EGFR</i> Rearrangements.","authors":"Grégoire Cullot,&nbsp;Samuel Amintas,&nbsp;Laura Karembé,&nbsp;Valérie Prouzet-Mauléon,&nbsp;Julie Rébillard,&nbsp;Lisa Boureau,&nbsp;David Cappellen,&nbsp;Aurélie Bedel,&nbsp;François Moreau-Gaudry,&nbsp;Stéphanie Dulucq,&nbsp;Sandrine Dabernat,&nbsp;Béatrice Turcq","doi":"10.1089/crispr.2022.0070","DOIUrl":"https://doi.org/10.1089/crispr.2022.0070","url":null,"abstract":"<p><p>Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of <i>BCR::ABL1</i> mRNA, a hallmark of chronic myeloid leukemia (CML), and <i>EGFR</i> DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of <i>BCR::ABL1</i> and <i>EGFR</i> alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.</p>","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9699854","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiplex Editing of the Nucleoredoxin1 Tandem Array in Poplar: From Small Indels to Translocations and Complex Inversions","authors":"Yen-Ho Chen, Shakuntala Sharma, W. P. Bewg, Liang-Jiao Xue, Cole R. Gizelbach, Chung-Jui Tsai","doi":"10.1101/2022.08.26.505498","DOIUrl":"https://doi.org/10.1101/2022.08.26.505498","url":null,"abstract":"The CRISPR-Cas9 system has been deployed for precision mutagenesis in an ever-growing number of species, including agricultural crops and forest trees. Its application to closely linked genes with extremely high sequence similarities has been less explored. Here, we used CRISPR-Cas9 to mutagenize a tandem array of seven Nucleoredoxin1 (NRX1) genes spanning ~100 kb in Populus tremula × alba. We demonstrated efficient multiplex editing with one single gRNA in 42 transgenic lines. The mutation profiles ranged from small indels and local deletions in individual genes to large genomic dropouts and rearrangements spanning tandem genes. We also detected complex rearrangements including translocations and inversions resulting from multiple cleavage and repair events. Target capture sequencing was instrumental for unbiased assessments of repair outcomes to reconstruct unusual mutant alleles. The work highlights the power of CRISPR-Cas9 for multiplex editing of tandemly duplicated genes to generate diverse mutants with structural and copy number variations to aid functional characterization.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49032740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR Crops and Sustainable Agriculture.","authors":"Rodolphe Barrangou","doi":"10.1089/crispr.2023.0002.editorial","DOIUrl":"https://doi.org/10.1089/crispr.2023.0002.editorial","url":null,"abstract":"","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9334493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}